Yang et al. BMC Complementary and Alternative Medicine 2012, 12:250 http://www.biomedcentral.com/1472-6882/12/250

RESEARCH ARTICLE Open Access Inhibition of -induced nitric and prostaglandin E2 production by fraction of Cudrania tricuspidata in RAW 264.7 Gabsik Yang, Kyungjin Lee, Mihwa Lee, Inhye Ham and Ho-Young Choi*

Abstract Background: Cudrania tricuspidata extract is an important traditional herbal remedy for tumors, , gastritis, and liver damage and is predominantly used in Korea, China, and Japan. However, the anti-inflammatory effects of the extract have not yet been conclusively proved.

Methods: In this study, we investigated the effects of the CHCl3 fraction (CTC) of a extract of C. tricuspidata on the lipopolysaccharide (LPS)-induced (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 cells and mouse peritoneal macrophages, and the levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in RAW 264.7 macrophage cells. Results: We observed that the protein expression levels of inducible NO synthase and COX-2 were markedly inhibited by CTC in a concentration-dependent manner. In addition, CTC reduced the production of TNF-α, IL-1β, and IL-6 in the LPS-stimulated RAW 264.7 macrophage cells. Conclusions: Our results show that the C. tricuspidata extract could modulate macrophage-mediated inflammatory functions such as the overproduction of cytokines, NO, and PGE2. The CTC was found to be the active fraction in this context.

Keywords: Cudrania tricuspidata, Nitric oxide, Prostaglandin E2, Lipopolysaccharide CHCl3 partitioned methanol extract of Cudrania tricuspidata, CTC, Lipopolysaccharide, LPS, prostaglandin E2, PGE2, nitric oxide, NO

Background Macrophages are extraordinarily versatile cells, and Inflammation is the result of the host’s immune response the ability of the macrophages to exploit their full func- to pathogenic challenges or tissue injuries, and under tional repertoire is essential for host immunity [3]. normal conditions, tissue structure and function are Macrophages detect pathogenic substances through restored to normal when the response ends. Moreover, pattern-recognition receptors and subsequently initiate normal inflammatory responses are self-limited by a and regulate inflammatory responses [3] by using a wide process that involves the downregulation of pro- range of soluble pro-inflammatory mediators. Lipopoly- inflammatory proteins and the upregulation of anti- saccharide (LPS) is one of the most powerful activators inflammatory proteins [1]. Thus, acute inflammation is of macrophages, and macrophages and monocytes that essentially a beneficial process, particularly in response have been activated by LPS are known to produce in- to infectious pathogens, whereas chronic inflammation flammatory mediators such as nitric oxide (NO) and is an undesirable phenomenon that can to inflam- other free radicals, in addition to numerous cytokines matory diseases [2]. such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 [4-6]. Bacterial pathogens, like LPS, * Correspondence: [email protected] activate cytokine networks by inducing many pro- Department of Herbology, College of Oriental Medicine, Kyung Hee inflammatory genes. Moreover, these inductions are University, 1 Hoegi-Dong, Seoul, Dongdaemun-Gu, Republic of Korea

© 2012 Yang et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Yang et al. BMC Complementary and Alternative Medicine 2012, 12:250 Page 2 of 7 http://www.biomedcentral.com/1472-6882/12/250

mediated via the activation of inducible transcription fac- dissolved in DMSO (Sigma, St. Louis, MO, USA) to ob- tors [7]. During the inflammatory process, large amounts tain a final concentration of 100 mg/ml, and was stored of the pro-inflammatory mediators NO and prostaglan- at −20°C until required. To use, the extract was further din E2 (PGE2) are generated by the inducible isoforms of diluted to different concentrations by using cell culture NO synthase (NOS) and cyclooxygenase-2 (COX-2) [8]. media (DMSO < 0.1%). In mammalian cells, NO is synthesized by 3 isoforms of NOS, namely, neuronal NOS (nNOS), endothelial NOS Animals (eNOS), and inducible NOS (iNOS). Although nNOS Male BALB/c mice (6–8 weeks of age) were purchased and eNOS are constitutively expressed, iNOS is specific- from the Korean branch of Taconic, Samtaco (Osan, ally expressed in response to interferon-γ (IFN-γ), LPS, Korea). Mice were kept in a temperature- and humidity- and various pro-inflammatory cytokines [9,10]. Several controlled, pathogen-free animal facility at Kyung Hee studies have suggested that the overexpression of COX-2 University and provided standard mouse chow and or iNOS is intimately involved in the pathogenesis of in- ad libitum. All animal care and experimental procedures flammation, cancer, , Parkinson’s syn- were complied with university guidelines of by Korean drome, Alzheimer’s disease [11], and other diseases. National Veterinary Research and Quarantine Service, Thus, many efforts have been made to develop World Organization of Animal Health (WOAH) and inhibitors or repressors of enzyme formation that select- were approved by the Ethical Committee for Animal ively target the inducible forms of these enzymes and, in Care and the Use of laboratory animals Kyung Hee particular, do not affect the activities of their respective University (Approval number # KHUASP(SE)-11-039). constitutive isoforms. Therefore, inhibiting the produc- tion of NO and PGE2 is an important therapeutic target Cell culture and sample treatment in the development of anti-inflammatory agents. RAW 264.7 cells were obtained from the Korean Cell Line Cudrania tricuspidata (CT) is a deciduous tree dis- Bank (Seoul), and were grown at 37°C in DMEM medium tributed across South Korea, China, and Japan, and the supplemented with 10% FBS, penicillin (100 units/ml), cortex and root bark of CT have been frequently used as and streptomycin sulfate (100 mg/ml) in a humidified 5% a traditional medicine to treat inflammation and tumors CO2 atmosphere. Cells were incubated with CT at increas- [12]. Several previous reports have described various ing concentrations (62, 125, 250, and 500 mg/ml), and effects of the CT extract, which include antioxidant ac- then stimulated with LPS (1 or 10 μg/ml) for the indicated tivity [13], inhibitory effect on NOS [14], and effects on times. the proliferation of inflammatory immune cells and tumor cells [15,16]. However, no studies to date have Macrophage isolation and culture investigated the effects of the CHCl3 fraction (CTC) of Mice were injected intraperitoneally (i.p.) with 2 ml the methanol extract of CT on inflammatory reactions. of sterile thioglycollate medium (Becton Dickinson, In the present study, we show that CTC reduces the Franklin Lakes, NJ, USA). Three days later, macrophages production of NO, PGE2, and TNF-α in RAW 264.7 were collected by peritoneal lavage with cold Dulbecco’s cells and mouse macrophage cells when these cells are modified Eagle’s medium (DMEM). After centrifugation treated with recombinant LPS. of the lavage, red blood cells were removed by lysis for 3 min in lysis buffer (150 mM NH4Cl, 1 mM KHCO3, Methods and 0.1 mM Na2EDTA). After centrifugation, the cells Plant materials were resuspended in DMEM supplemented with 1% Non-cultivated CT was collected from the Chiri Moun- antibiotic and antimycotic solution (Invitrogen, Carls- tain in the far south of the Republic of Korea. Collection bad, CA, USA) and 10% fetal bovine serum (WeIGENE, was made on February 20, 2007, a time when the plants Deagu, Korea), then incubated for 90 min in a humidi- have many thorns and few leaves. The plant materials fied atmosphere of 5% CO2 at 37°C. Non-adherent cells were identified by Professor Ho-Young Choi at the Her- were removed by washing, and the adherent cells were bology Laboratory, College of Oriental Medicine, Kyun- harvested and seeded for the various assays. ghee University, Seoul. Fresh CT was removed using a sickle and dried in a dark, well-ventilated place. Air-dried MTS-tetrazolium salt assay CT (1 kg) was cut into pieces by using a fodder chopper, Cell viability was measured based on the formation of and the extract was prepared by incubating with 80% blue formazan metabolized from colorless MTS by mito- methanol at room temperature for several days. Then the chondrial dehydrogenases, which are active only in live CT methanol extract (CTM) was fractionated using cells. RAW 264.7 macrophages were plated in 96-well 3 CHCl3, and the CHCl3 extracts were evaporated to afford plates at a of 4 × 10 cells per well for 24 h, and a CHCl3-soluble (14 g) fraction (CTC), which was then washed. Cells incubated with various concentrations Yang et al. BMC Complementary and Alternative Medicine 2012, 12:250 Page 3 of 7 http://www.biomedcentral.com/1472-6882/12/250

of CTM and CTC were treated with LPS (E. coli 026:B6; Measurement of PGE2 levels Sigma, MO, USA) for 24 h and then incubated in PGE2 levels in culture media were analyzed using PGE2 0.5 mg/ml MTS solution. Viabilities were determined assay kits (ParameterTM; R&D Systems, MN, USA). The using colorimetric MTS assay. PGE2 standard and the RD5-39 in the kits were used to construct a standard curve. Briefly, 100 μl of the Determinations of concentrations medium was mixed with 50 μl of primary antibody so- 5 RAW 264.7 cells were plated at 2.5 × 10 cells/ml in 24 lution and PGE2 conjugate and incubated for 2 h at well-plates and then incubated with or without LPS room temperature on a shaker. Cells were then washed (1 μg/ml) in the absence or presence of various concen- out of wells using 400 μl of 1× washing buffer; then, trations (62, 125, 250 and 500 μg/ml) of CTC for 24 h. color reagent (200 μl) was added and, 30 min later, The nitrite accumulated in culture medium was mea- stop solution (50 μl) was added and mixed in. Absorb- sured as an indicated of NO production based on the ance was measured at 450/570 nm using a VersaMaxTM Griess reaction. Briefly, 100 μl of cell culture medium microplate reader (Molecular Devices, CA, USA). was mixed with 100 μl of Griess reagent [equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid Cytokine array assays and 0.1% (w/v) naphtylethylenediamine-HCl], incubated Mouse cytokine antibody array membranes coated with at room temperature for 10 min, and then the absorb- 40 specific cytokine antibodies (RayBioTM Mouse In- ance at 540 nm was measured in a microplate reader flammation Antibody Array 1, RayBiotech, Inc.) were (VersaMax™, Molecular Device, USA). Fresh culture probed with isolated total protein samples. The mem- medium was used as the blank in all experiments. The branes were blocked by incubation in blocking buffer for amount of nitrite in the samples was measured with the 30 min at room temperature and then incubated with serial dilution standard curve of nitrite. samples for 1 h at room temperature. Membranes were then washed three times with Wash Buffer I and twice Western blot analysis with Wash Buffer II for 5 min at room temperature per Cellular proteins were extracted from control and CT- wash and then incubated with biotin-conjugated anti- treated RAW 264.7 cells. Briefly, cells were collected by bodies at 4°C overnight. Membranes were then washed, centrifugation and the pellets so obtained were washed incubated with HRP-conjugated streptavidin for 1 h at once with phosphate-buffered saline (PBS). Pellets were room temperature, incubated with detection buffer for then resuspended in extraction lysis buffer (50 mM 1 min, and exposed to X-ray film for 40 s (Kodak Inc.). HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1% Noni- Exposed films were digitized and relative cytokine levels det P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM were determined by densitometry (Scion NIH Image dithiothreitol, 5 mM Na fluoride and 0.5 mM Na ortho- 1.63), by subtracting background staining level and nor- vanadate) containing 5 mg/ml leupeptin and 5 mg/ml malizing to positive control spots, which were used as aprotinin and incubated for 20 min at 4°C. Cell debris internal standards. was removed by microcentrifugation, and supernatants were rapidly frozen. Protein concentrations were deter- mined using the Bio-Rad protein assay reagent (Hercules, CA), according to the manufacturer’s instruc- tions. Proteins (40 μg) were separated by 10% SDS- polyacrylamide gel electrophoresis, and electroblotted onto nitrocellulose membranes. Immunoblots were incu- bated overnight with blocking solution (5% skimmed milk) at 4°C, and then for 4 h with a primary antibody. Blots were then washed three times with Tween 20/Tris- buffered saline (TBST), incubated in 1:100 alkaline phosphatase-conjugated secondary antibody for 1 h at room temperature, washed three times with TBST, and finally reacted with BCIP-NBT solution (Nakanai Tesque, Japan). The monoclonal antibodies (iNOS, COX-2 and β-actin), and the peroxidase-conjugated sec- Figure 1 Effect of CTM on the viability of LPS-stimulated RAW ondary antibody were purchased from Santa Cruz Bio- 264.7 cells. Cells (5 × 104) were incubated with CTM and CTC technology, Inc. (Santa Cruz, CA, USA). Density ratios (62, 125, 250, 500 μg/ml) at 37°C for 24 h. Cell viabilities were versus β-actin were determined by densitometry analysis evaluated using MTS assays. Data represent the means ± SDs of three independent experiments. (Scion NIH Image 1.63). Yang et al. BMC Complementary and Alternative Medicine 2012, 12:250 Page 4 of 7 http://www.biomedcentral.com/1472-6882/12/250

Figure 2 Effect of CTM on LPS-induced NO levels in RAW 264.7 cells. (A) Cells were treated with different concentrations of CTM and CTC (62, 125, 250, 500 μg/ml) for 1 h, then with LPS (10 μg/ml) for 24 h. Culture supernatants were subsequently isolated and analyzed for nitrite levels. (B) Nitrite levels in culture supernatants were measured after macrophages (2 × 106 cells/ml) were primed with IFN-γ (0.5 ng/ml) for 2 h and then stimulated with LPS (1 μg/ml) in the presence of CTM and CTC for 18 h. Values are means ± SDs of three separate experiments. (#p < 0.05 vs. the non-treated control group; *p < 0.05, **p < 0.01 vs. the LPS-treated group).

Figure 3 Effect of CTM on LPS-induced PGE2 production in RAW 264.7 cells. (A) PGE2 production was measured in RAW 264.7 cells treated with different concentrations of CTM and CTC (62, 125, 250, 500 μg/ml) for 1 h, then incubated with LPS (10 μg/ml) for 24 h. (B) PGE2 production in culture supernatants was measured after macrophages (2 × 106 cells/ml) were primed with IFN-γ (0.5 ng/ml) for 2 h and then stimulated with LPS (1 μg/ml) in the presence of CTM and CTC for 18 h. Values are means ± SDs of three separate experiments. (#p < 0.05 vs. the non-treated control group; *p < 0.05, **p < 0.01 vs. the LPS-treated group). Yang et al. BMC Complementary and Alternative Medicine 2012, 12:250 Page 5 of 7 http://www.biomedcentral.com/1472-6882/12/250

Statistical analysis Effects of CTC on LPS-induced NO and PGE2 production The results shown were obtained from at least three in- To investigate the anti-inflammatory properties of dependent experiments and are presented as means ± CTC, we used RAW 264.7 cells, which produce NO SDs. Statistical analyses were performed by one-way ana- and PGE2 when stimulated by LPS. The cells were pre- lysis of variance (ANOVA) with Tukey’s and Duncan’s incubated with varying concentrations of CTM and post hoc tests. All statistical analyses were performed CTC (each at 62 μg/ml, 125 μg/ml, 250 μg/ml, and using SPSS v12.0 software. P values < 0.05 were consid- 500 μg/ml) for 1 h and then stimulated with 10 μg/ml ered to indicate statistical significance. LPS for 24 h. LPS and CT were not added to the controls (Con). The nitrite and PGE2 levels in the media were determined, and we observed that CTC Results reduced NO and PGE2 production in a dose- Effects of CTC on the viability of RAW 265.7 cells dependent manner (Figures 2A and 3A). To examine We examined the effects of CTM on the viability of the potential effects of CTC on LPS-induced NO and RAW 264.7 cells in vitro by incubating cells with 62 μg/ PGE2 production in macrophages, the cells were pre- ml, 125 μg/ml, 250 μg/ml, or 500 μg/ml of CT or CTC treated with IFN-γ (0.5 ng/ml) for 2 h and then for 24 h. The results of the MTS assay showed that CT stimulated with LPS (1 μg/ml) and varying concentra- and CTC were not cytotoxic to RAW 264.7 cells at con- tions of either CTM or CTC (62 μg/ml, 125 μg/ml, centrations up to 500 μg/ml (Figure 1). 250 μg/ml, and 500 μg/ml each) for 18 h. The NO

Figure 4 Effect of CTC on LPS-induced iNOS and COX-2 protein levels in RAW 264.7 cells. (A) Cells were treated with different concentrations of CTC (125, 250, 500 μg/ml) for 1 h and then with LPS (10 μg/ml) for 24 h. β-actin expression is shown as a loading control. (B) Relative expression of iNOS and COX-2. Bands on western blots were quantified by densitometry. The values are expressed as the means ± SD of at least three independent experiments (#p < 0.05 vs. the non-treated control group; *p < 0.05, **p < 0.01 vs. the LPS-treated group). Yang et al. BMC Complementary and Alternative Medicine 2012, 12:250 Page 6 of 7 http://www.biomedcentral.com/1472-6882/12/250

and PGE2 concentrations in the culture supernatants Discussion were suppressed by CTC in a dose-dependent manner The present study was undertaken to elucidate the (Figures 2B and 3B). pharmacological and biological effects of CTC on the production of inflammatory mediators in RAW 264.7 macrophages. The results indicate that CTC is an effect- Effects of CTC on LPS-induced iNOS and COX-2 protein ive inhibitor of LPS-induced NO, PGE2, TNF-α, IL-1β, Levels and IL-6 production in these cells. Furthermore, these Western blotting was performed to determine whether the inhibitory effects of CTC were accompanied by dose- inhibitory effects of CTC on NO and PGE2 production dependent decreases in the protein levels of iNOS and were related to the changes in the expressions of iNOS COX-2. These findings indicate that the expression of and COX-2. LPS markedly upregulated iNOS and COX- iNOS and COX-2 in RAW 264.7 cells is suppressed by 2 protein levels, and pre-treatment with CTC inhibited CTC, and that this suppression to the downregula- these upregulations (Figure 4). These results indicate that tion of NO and PGE2. the inhibitory effects of CTC on LPS-induced NO and NO is a key inflammatory mediator, and excessive NO PGE2 production are due to the suppression of iNOS production occurs in both acute and chronic inflamma- and COX-2. tion. NO is synthesized from L- by NOS isoen- zymes, one of which is iNOS, which is expressed Effects of CTC on the LPS-induced production of TNF-α, predominantly in activated macrophages [17]. Similarly, IL-1β, and IL-6 PGE2 is a well-known inflammatory mediator produced The effects of CTC on LPS-induced TNF-α, IL-1β, and by the action of COX-1 and COX-2 on arachidonic acid. IL-6 release were investigated using a cytokine array. We COX-2 expression is induced by cytokines, LPS, and observed that pretreatment of RAW 264.7 cells with other activators during inflammation, resulting in the CTC (250 μg/ml) decreased the TNF-α, IL-1β, and IL-6 release of high levels of PGE2 at the sites of inflammation production (Figure 5). [18]. In this study, we showed that in LPS-stimulated

Figure 5 Effect of CTC on LPS-induced cytokine expression in RAW 264.7 cells. Levels of the cytokines TNF-α, IL-1β and IL-6 were monitored 24 h after treating cells with LPS (10 μg/ml) with or without CTC. Yang et al. BMC Complementary and Alternative Medicine 2012, 12:250 Page 7 of 7 http://www.biomedcentral.com/1472-6882/12/250

macrophages, CTC dose-dependently inhibited NO and Received: 13 March 2012 Accepted: 24 November 2012 PGE2 production. The levels of iNOS and COX-2 were Published: 10 December 2012 also downregulated by CTC, suggesting that this was the References mechanism underlying the observed suppression of NO 1. Lawrence T, Willoughby DA, Gilroy DW: Anti-inflammatory mediators and PGE2 production. and insights into the resolution of inflammation. Nat Rev Immunol 2002, 2(10):787–795. LPS stimulates macrophages to produce iNOS, COX-2 2. Kaplanski G, et al: IL-6: a regulator of the transition from neutrophil to and pro-inflammatory cytokines such as TNF-α, IL-1β, monocyte recruitment during inflammation. Trends Immunol 2003, and IL-6 [17]. In addition, these pro-inflammatory med- 24(1):25–29. 3. Medzhitov R, Janeway CA Jr: Innate immunity: the virtues of a nonclonal iators play important roles in the pathogenesis of various system of recognition. 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CHCl3: Partition of the methanol extract of Cudrania tricuspidata; CTC: Lipopolysaccharide; LPS: Prostaglandin E2; PGE2: Nitric oxide; doi:10.1186/1472-6882-12-250 NO: Inducible isoform of NO synthase; iNOS: Cyclooxygenase-2 COX-2. Cite this article as: Yang et al.: Inhibition of lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by chloroform fraction of Cudrania tricuspidata in RAW 264.7 macrophages. BMC Complementary Competing interests and Alternative Medicine 2012 12:250. The authors declare that they have no competing interests.

Authors’ contributions GY carried out the in vitro studies and drafted the manuscript. GY and ML carried out the immunoassays. HC and IH participated in the design of the study and performed the statistical analysis. KL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Acknowledgement This work was supported by the Second Stage of the Brain Korea 21 project in the Oriental Medical Science Center.